| Abstract:Biomining is a microbe-mediated engineering process that has been use in these last decades to extract some base and precious metals from mineral ores and concentrates. Biomining is a general term that can be divided into two different techniques:bioleaching and bio-oxidation. The term bioleaching refers to the conversion of an insoluble metal (usually a metal sulfide, e.g., CuS, NiS, and ZnS) into a soluble form (usually the metal sulfate, e.g., CuSO4, NiSO4, ZnSO4) and a subsequent extraction of the desired metal (Cu, Ni, Zn) from the solution. In the case of bio-oxidation, the desired metal (e.g., gold, silver) is not solubilized, so the activity of leaching bacteria is applied only to remove interfering metal sulfides from ores bearing the precious metals prior to cyanidation treatment. In general the treated ores are in two forms:metal oxides (in case of uranium) and metal sulfides ores, the latter being the most important one in terms of quantity. The metal sulfides are also categorized into two groups according to their acid solubility:acid-soluble and acid-insoluble metal sulfides. Huge efforts have been made in investigating the mechanisms involved in the bio-oxidation of these two types of metal sulfides, and outstanding developments have been achieved, even though some aspects are controversial and still remain open questions.Two pathways have been proposed for metal sulfides dissolution: thiosulfate pathway (for acid-insoluble mineral sulfides) and polysulfide pathways (for acid-soluble minerals sulfides). For both of these pathways, leaching microorganisms intervene by providing the main leaching agents that are ferric iron and proton H+. Most of these microorganisms are prokaryotes (bacteria and archaea). They present different physiological and biochemical characteristics and interact between them. They are mostly classified according to their optimal temperature for growth. Thus, three groups are found in leaching environment:mesophiles (mostly constituted of gram negative bacteria), moderate thermophile (constituted of archaea and gram positive bacteria), and thermophiles (mostly constituted of archaea). Because ferric ions and in some extent proton H+are the relevant agents for metal sulfides dissolution, the suppliers iron-oxidizer (ferric ions suppliers) and sulfur oxidizers (proton H+suppliers) are considered the main leaching microorganisms. However, they share their habitat with a third microbial group (heterotrophs). They include prokaryotes (bacteria and archaea) and eukaryotes (fungi). The heterotrophic acidophiles do not intervene directly in bioleaching, but are believed to impact on leaching efficiency, since they carry on positive or negative effects on the main leaching microbes. One of their most important activities in bioleaching systems is that consisting to scavenge low molecular weight organic matter such as organic acids known to be highly toxic to acidophilic iron-and sulfur-oxidizing bacteria. This activity on carbon cycling has been demonstrated to positively impact on bioleaching efficiency since they strengthen and increase the viability of leaching microbial community known to be highly sensitive to dissolved organic matter (DOM). Another contribution of acidophilic heterotrophs in bioleaching is the production of CO2and ferrous irons which are used by autotrophs as carbon and energy sources, respectively. Heterotrophs have been also found to participate in the establishment of the biofilm (Extracellular Polymorphic Substance+microorganisms) considered as a biogenic reaction space in which metal dissolution reactions occur.In these last decades outstanding advances have been made in understanding the mechanisms underlying the microbial dissolutions of metal sulfides. These advances have led to the optimization of the existing bioleaching technologies and the implementation of new bioleaching approaches. However, like any bioprocess, bioleaching faces challenges that need to be thoroughly addressed in order to make it more competitive. One of those challenges is obviously finding stronger and more efficient microorganisms or microbial communities that can provide higher leaching performances and work in extreme conditions. However seeking for stronger microorganisms faces another challenge which is their cultivation and purification. In fact, as stated above, these microbes are so sensitive to dissolved organic matter causing enormous difficulties for their cultivation and purification, and consequently their studies.Keeping this view in mind, we have proposed to develop an new cultivation method based on microbe-microbe interaction to isolate and/or purify leaching strains, study the leaching efficiency of native and foreign microorganisms through the cross-comparison of strains from very different and distant copper mining sites:Chambishi copper mine (Zambia, Africa) and Dexing copper mine (China, Asia). The bioleaching isolates were also tested under various stress condition including, pH, temperature, heavy metals, dissolved organic matter. For organic acids stress the experiment were conducted in presence or absence of a new isolated yeast strains to study the impact of the latter on the viability of leaching bacteria, and its subsequent influence on leaching efficiency.The objectives of the study were:(1) Development of an efficient and reproducible cultivation technique for a rapid isolation, purification and enumeration of acidophilic iron-and sulfur-oxidizing microorganisms.(2) Study interactions that may exist between leaching bacteria and acidophilic yeast strains in bioleaching environment, and how the supposed interactions may affect acidophilic autotrophs viability and their leaching efficiency.(3) Comparison of indigenous and foreign leaching microorganisms in terms of physiological and biochemical characteristics as well as leaching efficiency.For the purpose of this work, sample taken from a heap leaching plant located in Zambia (Southern Africa) was used to isolate leaching strains. Altogether three leaching strains and one yeast strain were isolated. The yeast strain was isolated on a single layer agar plate supplemented with2.5mM of tetrathionate and0.025%wt/v of TSB (tryptone soya broth), and solidified with1.5%wt/v agar. The new isolated yeast strain was incorporated in the gel under layer of a double-layer agar plate for the isolation of acidophilic iron and sulfur oxidizers. The double layer medium was prepared as follows:A mixed solutions at50℃with a final concentration of1.5%(wt/v) agar,25mM ferrous iron,2.5mM tetrathionate and0.025%wt/v TSB was prepared. The combined medium was immediately inoculated with the new isolated yeast strain (0.5-1%v/v), and poured as thin gels in sterile petri plates. Once these have solidified, a top layer of sterile medium was added.Once the cultures have been purified, the identification of the strains was performed through molecular phylogenetic analysis based on the18S rRNA gene and Internal Transcribed Spacer region (Comprising ITS1region,5.8S gene and ITS2region) for yeast species, and16S rRNA and gyrB genes for leaching bacteria. The results revealed that the yeast belongs to Candida digboiensis sp., while the leaching bacteria belong to Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferriphilum species. They were designated C. digboiensis NB, At. ferrooxidans FOX1, At. thiooxidans ZMB and L. ferriphilum BN. Other bacteria that belong to these three species were also used in the course of this study. They were isolated from Dexing copper mine, and are designated At. ferrooxidans YTW, At. thiooxidans A02and L. ferriphilum YTW315.All these microbial strains were tested under normal and various stress conditions including temperature (25-45℃); pH (1.2-3) for bacteria and (1.5-8) for the yeast strain; heavy metals such as ferric ions (0-600mM) and copper (0-70mM). Their carbon and energy sources were determined by culturing them in various organic compounds (glucose, sucrose, tryptone, peptone, acetic acid, propionic acid) and inorganic product (ferrous sulfate, elemental sulfur, tetrathionate, thiosulfate, metal sulfides).Influence of heterotrophic yeast strain C. digboiensis on leaching bacteria growth and activity was also studied. Bacterial cells were cultivated in9K medium supplemented with ferrous (25mM) and elemental sulfur (1%wt/v);10mM of acetic and propionic acid was also added. Two systems were made up: those inoculated with leaching bacteria and yeast strain (referred to as co-inoculation system) and those inoculated with leaching bacteria only (referred to as control system). Growth rates, iron and sulfur oxidation rates as well as organic acid consumption rates were determined to monitor this experiment.The implication of yeast EPS in improving iron and sulfur oxidation rate was also studied. For this experiment the iron-and sulfur-oxidizers were co-inoculated with EPS-free or untreated yeast culture (with EPS yeast culture) in9K medium supplemented with ferrous iron or elemental sulfur. Iron and sulfur oxidation rates in different systems were compared to determine the impact of the yeast EPS.The yeast-bacteria interactions were also studied in bioleaching experiment. Minerals sample was taken from Chambishi copper mine. Three systems were set up to showcase the effect of C. digboiensis NB on leaching bacteria viability, and consequently on leaching efficiency. One system was inoculated with leaching bacteria and viable yeast strain (referred to as co- inoculation system). The second system was inoculated with leaching bacteria only (referred to as test control system). The last one was abiotic, bacterial culture was replaced with an equivalent volume of9K medium (referred to as abiotic control system). Bioleaching parameters including pH, Eh, microbial growth, iron and copper extraction rate as well as content of dissolved organic matter were determined at regular time to reveal in one hand the interactions between the two microbial groups; and in the other hand the contribution of the heterotroph on microbial community leaching efficiency.Similar experiment was carrying out using pyrite-laden acetic acid. The objective of this experiment was to simulate the role of the yeast strain in sludge bioleaching, which usually contents high level of dissolved organic matter (up to45%organic matter). The experiment was conducted as above; only10mM of acetic acid was added in the bioleaching systems.The last and the most important part of this work consisted of comparing foreign and native leaching bacteria for the bioleaching of low grade copper ores. Bacteria isolated from Chambishi copper mine (Zambia, Africa) and Dexing copper mine (China, Asia) were studied for the bioleaching of their native ores (mineral sample from Dexing and Chambishi copper mine). In bioleaching of the Dexing mineral sample, Dexing strains and Zambia strains were considered as native and foreign microbial community, respectively; and vice versa. This experiment was carried out in pure and mixed culture to showcase the contribution of each strain. In addition to the parameters determined in the above bioleaching experiments, microbial community composition and leaching residues mineral composition were also determined.In terms of results, the isolated yeast strain as the other members of C. digboiensis species was found to be able to utilize various organic compounds as carbon and energy sources including organic acids. These characteristics together with its tolerance to pH (growth occurs in pH ranging from pH1.5to pH8.0) confer the yeast strain the properties to scavenge organic matter initially present in agar or produced during incubation, and consequently allow a rapid and efficient growth of acidophilic autotrophs on agar plate, no matter the quality of the gelling agent. The use of the yeast strain, due to its high metabolic activity, shortened the growth of leaching bacteria from weeks to4-5days. In addition, unlike heterotrophic prokaryotes such as Acidiphilium sp. and Acidocella sp. the yeast strain presented a high tolerance to tetrathionate, which its combination with ferrous ions allowed the growth of different leaching bacteria and their differentiation according to colonies’morphologies.Altogether three leaching strains were isolated from Zambia, and three other (previously isolated from Dexing) purified using this modified double-layer technique. Except copper tolerance, strains from the same species presented almost the same physiological characteristics. They all presented interesting potentials for their use in bioleaching, their optimal pH were below pH2.0and their optimal temperatures for growth ranged between30and35℃. Furthermore they could all generate ferric ions and/or protons H+through the bio-oxidation of ferrous ions and sulfur compounds, respectively. They presented also high tolerance to ferric ions and copper known as the main inhibitors in bioleaching of copper sulfides ores. None of them could use organic compound, but they were differently affected by dissolved organic matter.About organic matter, as expected, the isolated leaching bacteria were found very sensitive to organic acid such as acetic and propionic acids. However, C. digboiensis NB was found to increase the viability and activity of the autotrophs under organic acid stress, and consequently to enable their growth under such conditions. The yeast strain utilized the organic acids compounds as sole energy and carbon source, and subsequently created suitable conditions for growth and activity of the new isolated leaching acidophiles. This result together with that obtained with the double-layer plate strongly suggests that the fact that these two microbial groups (autotrophic leaching bacteria and heterotrophic yeast strain) share the same habitat is not fortuitous; it follows mutualistic interactions occurring between them. This assumption was further confirmed by the bioleaching of low-grade copper mineral sulfide sample with the new leaching strains, and in presence or absence of the yeast strain. The leaching efficiency increased by8.7%with the introduction of C. digboiensis NB. In fact the latter utilized organic compounds produced by autotrophs, and as such avoided its accumulation in leaching system, which in some extent promote the viability and the leaching efficiency of acidophilic iron-and sulfur-oxidizing bacteria. Beside the degradation of organic matter, the yeast EPS was found to possess surfactant activity, and as such could change the surface tension between the sulfur and the liquid medium, thus facilitating the attachment of sulfur oxidizers on sulfur particles. This phenomenon points out another contribution of yeast strain in enhancing leaching ability of autotrophs.The impact of the yeast strain was more significant for the bioleaching of organic acid-laden-pyrite. This experiment was done in order to simulate the role of yeast strain in bioleaching of sludge-laden heavy metals. In this experiment, the percentage of total iron extracted in control (leaching bacteria only) and co-inoculation (leaching bacteria and yeast strain) systems, after24days of bioleaching, was6.1%and82.3%, respectively. This finding proved that C. digboiensis coupled with autotrophic leaching bacteria could be successfully employed for the removal of heavy metals from sludge prior to land application.Leaching strains for a given species presented almost the same physiological characteristics and potentials in bioleaching with slight advantages of Zambian leaching bacteria. Surprisingly, they presented different activity according to their status (native or exogenous). Native microbial communities were found more active than exogenous microorganisms. In instance the Dexing strains showed higher cell density for the bioleaching of the Dexing mineral sample in comparison with Zambian isolates, and vice versa for the Chambishi mineral sample. And this was reflected in the leaching efficiency; the Zambia and Dexing consortia achieved copper recovery of89%and84%for the bioleaching of the Zambian mineral sample, respectively. These results implied that physiological and biochemical characteristics are necessary conditions for the use of a leaching strain in bioleaching, but not sufficient to provide higher leaching efficiency. In complex bioleaching systems, various factors come to account; and the leaching potential of the microorganisms is just one of them. The use of native microorganisms can be an interesting alternative to overcome the ineffectiveness of available commercial leaching strains for the bioleaching of new sulfides mineral ores.In summary,(1) A new yeast strain was isolated from a heap leaching plant located at Chambishi copper mine (Zambia, Africa). Based on its ability to utilize various organic compounds, the yeast strain was used in a lower layer of double-layer agar plate to effectively isolate and purify leaching acidophiles. (2) The yeast strain could enable also the growth of iron-and sulfur-oxidizing bacteria under organic acid stress.(3) Also, by avoiding the accumulation of toxic dissolved organic matter, and eliminating the organic compound initially present in the leaching system, the yeast strain could contribute somehow to the leaching efficiency of microbial leaching community.(4) This contribution was more significant in bioleaching of "sludge"(acetic acid-laden pyrite in this case).(5) Its EPS was also found to facilitate the attachment of microbial strains on hydrophobic particles such as sulfur particles.(6) Finally the comparison of the strains from Chambishi and Dexing copper mines revealed that native microbial community would give a better leaching efficiency than exogenous one, and as such their use may be an alternative to overcome the ineffectiveness of commercial leaching strains for the bioleaching of new ores. |
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